Familial Mediterranean fever (FMF) is characterized by episodes of fever with abdominal pain, pleurisy, and/or arthritis; some patients also develop systemic amyloidosis, leading eventually to renal failure. FMF is inherited as a single autosomal recessive gene (designated MEF), which is very common among Jewish, Armenian, Turkish, and Arab populations. Since the biochemical lesion in FMF is unknown, our goal is to identify the FMF gene by positional cloning. By the beginning of this year, we had narrowed the candidate interval to a region of approximately 1 Mb on the short arm of chromosome 16. In 1994-95 our efforts were divided among a) genomic cloning of the candidate interval; a) further genetic refinement of the interval; c) transcript identification; and d) functional studies of FMF leukocytes. Genomic cloning began with the identification of CEPH megaYACs. However, due to the instability of megaYACs from this part of the genome, a major effort in the last year has been directed towards devel-oping a cosmid contig. Using the Los Alamos 10X chromosome 16-specific cosmid library, we have con-structed a 525 kb contig on the telomeric end of the 1 Mb candidate interval, and a 350 kb contig on the centromeric end. We have made NotI and EcoRI restriction maps of these contigs. We are attempting to close the gap between the two contigs with a combination of cosmids, YACs, PACs, BACs, and P1s. Refined genetic mapping was facilitated by our identification of 4 new highly polymorphic microsatel-lites from the cloned genomic DNA. These new markers exclude an additional 250 kb on the telomeric end of the interval, and 300 kb on the centromeric end of the interval. Moreover, at one of the 4 marker loci there are no recombinants among a panel of 65 Jewish, Armenian, and Arab families. A specific allele of this latter locus is in strong linkage disequilibrium with FMF among North African Jewish FMF patients; Luria-Delbruck analysis of linkage disequilibrium data in the Moroccan Jewish founder popula-tion places MEF within about 80 kb of this marker. We are identifying transcripts from the FMF candidate interval by exon amplification from cosmids. To date, we have isolated a total of 28 putative exons from the region, and we are screening cDNA lib-raries for the corresponding full-length transcripts. In parallel, collaborators in Australia are using direct cDNA selection on cosmid pools to identify additional transcribed sequences. Phenotypic analysis of FMF leukocytes may facilitate analysis of positionally-defined candidate genes; over the last year we have confirmed that leukocytes from FMF patients show increased adhesion to fibronectin and type I collagen, but express normal levels of known cell-surface adhesion molecules.